CN115215953B - Self-assembled redox polymers, sensors and methods of making the same - Google Patents

Self-assembled redox polymers, sensors and methods of making the same Download PDF

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CN115215953B
CN115215953B CN202210837308.6A CN202210837308A CN115215953B CN 115215953 B CN115215953 B CN 115215953B CN 202210837308 A CN202210837308 A CN 202210837308A CN 115215953 B CN115215953 B CN 115215953B
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CN115215953A (en
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方凯
杨丽芬
李文俊
谭海兵
王娟
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Jiangxi Sitomai Medical Technology Co ltd
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Abstract

The invention relates to a self-assembled redox polymer, a sensor and a preparation method thereof. The self-assembled redox polymer has the following structure:wherein R is 1 And R is 2 Each independently represents a bond, and R 1 And R is 2 The same or different; m represents a transition metal; l (L) 1 、L 2 、L 3 、L 4 、L 5 And L 6 Each independently represents a ligand which is a heterocyclic ring and is coordinately bound to M through a heteroatom of the heterocyclic ring; x represents a self-assembled group; y represents an anion, and a represents an integer; b represents the number of anions and is a natural number; wherein b>a+1; n, m and r represent natural numbers, and the sum of n, m and r is more than 20; e. g and f represent integers. The self-assembled redox polymer of the invention can prepare the sensing layer of the sensor without introducing a cross-linking agent.

Description

Self-assembled redox polymers, sensors and methods of making the same
Technical Field
The invention relates to a self-assembled redox polymer, a sensor and a preparation method thereof, belonging to the technical field of medical diagnosis.
Background
With increasing standard of living, diabetic patients are also increasing year by year, early diagnosis, timely treatment, and continuous control are crucial to ensure quality of life for patients and to avoid circulatory problems caused by diabetes (such as renal failure, heart disease, and blindness). An enzymatic biosensor is a sensor that converts the biochemical reaction signal of an analyte into a measurable physical signal, such as a photoelectric signal, wherein the most studied biosensor is an amperometric glucose sensor.
The first generation of biosensing technology is to monitor glucose indirectly by detecting hydrogen peroxide or oxygen consumed during oxidation of glucose by electrochemical means. For example, guardian and iPro2 of Maidon force and Dexcom G5 and G6 of Dekang were developed based on the first generation biosensing technology, which monitors glucose by electrochemically detecting hydrogen peroxide generated by glucose oxidase during catalytic oxidation. Because the electrochemical method for detecting hydrogen peroxide has very strict requirements on electrodes, only very few materials such as uranium and uranium alloy can be used for manufacturing the glucose biosensor, the cost of the sensor of the implantable continuous glucose monitoring system is greatly increased. In addition, electrochemical detection of hydrogen peroxide requires a higher detection potential, thereby greatly reducing the anti-interference capability of the implantable continuous glucose monitoring system, particularly for commonly used antipyretics such as acetaminophen.
The second generation of biosensing technology is to implement direct electrochemical detection of glucose by introducing a redox mediator into the glucose biosensor. Unlike common protein molecules, glucose oxidase has a large molecular weight (160 kDa), its molecular structure, particularly the steric structure of the catalytic active center, is very complex, and is located inside glucose oxidase and deeply surrounded by various skin chains. Glucose oxidase cannot directly exchange electrons with the electrode. Heller et al (Acc. Chem. Res.23 (1990) 128-134) found that the incorporation of redox species redox mediators (redox small molecules such as iron fluorides or redox macromolecules) with which glucose oxidase can exchange electrons with electrodes was incorporated into glucose biosensors. However, because the redox mediator is a small molecule or a high molecular material, the preparation of the redox mediator is difficult to be controlled accurately, and meanwhile, the possibility that the redox mediator seeps out of the implantable glucose biosensor also exists, so that more uncertainty is brought to the performance of the implantable continuous glucose monitoring system.
The enzyme layer of the sensor in the current continuous blood glucose monitoring (CGM) in the market needs to be solidified by a cross-linking agent, such as glutaraldehyde, polyethylene glycol diglycidyl ether (PEGDGE) and the like, but the method has the advantages of slow cross-linking solidification, introduction of toxic small molecules which are easy to leak, and the risk of leakage of the unreacted complete cross-linking agent.
Citation 1 discloses an oxidoreductase having improved electrochemical activity and a biosensor comprising the same, which uses a chemical crosslinking method and is considered that chemically crosslinked glucose oxidase still maintains their direct electrochemical activity, for example, glutaraldehyde chemically crosslinked glucose biosensor comprising modified glucose oxidase exhibits good electrochemical performance on an electrode and is a typical surface electrochemical phenomenon. However, it does not take into account the risk of leakage of chemical crosslinking agents such as glutaraldehyde, which are slow to crosslink and cure.
Reference 2 discloses an electrochemical biosensor comprising carbon nanotubes for measuring biological signals and a method of manufacturing the same. The electrochemical biosensor for continuous glucose monitoring includes an electrode to which a sensing film including an oxidoreductase, an electron transfer medium, and a crosslinking agent is fixed together with a carbon nanotube, wherein the oxidoreductase oxidizes a target substance, and electrons thus generated during the oxidation are transferred through the electron transfer medium and the carbon nanotube. The electrochemical biosensor also does not consider the risk of leakage of chemical crosslinking agents such as glutaraldehyde and the like, which are slow to crosslink and solidify.
Citation literature:
citation 1: CN114152656A
Citation 2: CN114269246A
Disclosure of Invention
Problems to be solved by the invention
In view of the technical problems in the prior art, for example: the invention firstly provides a self-assembled redox polymer and a preparation method thereof, wherein the self-assembled redox polymer can prepare a sensing layer of a sensor on the premise of not introducing a cross-linking agent.
The invention also provides a sensor which has better electrochemical performance, can respond to the concentration of glucose, and promotes the high detection sensitivity, rapidness, accuracy, good stability and good repeatability of the biosensor.
Furthermore, the invention also provides a preparation method of the sensor, which is simple and easy to implement, raw materials are easy to obtain, and the prepared sensing layer does not need to introduce an additional small molecule cross-linking agent, is easy to operate, and can avoid the problem of cross-linking agent leakage.
Solution for solving the problem
The present invention first provides a self-assembled redox polymer having the following structure:
wherein R is 1 And R is 2 Each independently represents a bond, and R 1 And R is 2 The same or different;
m represents a transition metal;
L 1 、L 2 、L 3 、L 4 、L 5 and L 6 Each independently represents a ligand which is a heterocyclic ring and is coordinately bound to M through a heteroatom of the heterocyclic ring;
x represents a self-assembled group;
y represents an anion and is preferably selected from the group consisting of,
a represents an integer; b represents the number of anions and is a natural number; wherein b > a+1;
n, m and r represent natural numbers, and the sum of n, m and r is more than 20;
e. g and f represent integers.
The self-assembled redox polymer according to the present invention, wherein the self-assembled group is selected from one of the following substituted or unsubstituted substituents:
wherein c and d are integers from 1 to 16.
The self-assembled redox polymer according to the present invention wherein the substituents of the self-assembled groups are selected from benzyl bromide, benzyl chloride, -F, -Cl, -Br, -I, -NO 2 、-COOH、-SO 3 H、-NH 2 -OH, an alkoxy group having 1 to 6 carbon atoms or an alkyl group having 1 to 6 carbon atoms.
The self-assembled redox polymer according to the present invention has a cleavage molecular weight of 1000Da or more.
The invention also provides a preparation method of the self-assembled redox polymer, which comprises the step of grafting self-assembled groups on the redox polymer, preferably, the grafting mode comprises one or more than two of electrophilic reaction, nucleophilic reaction and condensation reaction.
The present invention also provides a sensor comprising: a flexible electrode comprising a substrate, a working electrode, a counter electrode, and a reference electrode, wherein,
the working electrode and the counter electrode are respectively arranged at two sides of the base material, and a dielectric layer is arranged at one side of the counter electrode far away from the base material;
a reference electrode is arranged on one side of the working electrode far away from the base material, a dielectric layer is arranged between the working electrode and the reference electrode, a dielectric layer is arranged on one side of the reference electrode far away from the working electrode, and,
the surface of the working electrode is provided with an exposed part without a dielectric layer and a reference electrode, and the surface of the exposed part is loaded with a self-assembled sensing layer, wherein the self-assembled sensing layer is derived from the self-assembled redox polymer and the redox enzyme.
The sensor according to the present invention, wherein the mass ratio of the self-assembled redox polymer and the redox enzyme is 1:0.5 to 1:2.
The sensor according to the present invention, wherein the surface of the flexible electrode has a cationic polymer; preferably, the cationic polymer has a weight average molecular weight of from 1000 to 50000Da.
The sensor according to the present invention, wherein a diffusion limiting membrane is further provided on the surface of the flexible electrode having the self-assembled sensing layer, preferably, the diffusion limiting membrane is derived from a polymer containing a nitrogen-containing heterocyclic group and a crosslinking agent; more preferably, the polymer containing nitrogen heterocyclic groups is modified with hydrophilic groups.
The invention also provides a preparation method of the sensor, which comprises the following steps:
a step of preparing a flexible electrode, wherein the flexible electrode comprises a substrate, a working electrode, a counter electrode and a reference electrode;
and loading the self-assembled sensing layer on the exposed part of the working electrode, which is not provided with the dielectric layer and the reference electrode.
According to the preparation method, after the self-assembled redox polymer and the redox enzyme are mixed, the self-assembled redox polymer and the redox enzyme are loaded on the surface of the exposed part of the working electrode, where the dielectric layer and the reference electrode are not arranged, so that the flexible electrode with the self-assembled sensing layer is obtained.
According to the preparation method, before the self-assembled sensing layer is loaded, the cationic polymer is initiated and grafted on the surface of the flexible electrode by utilizing plasma, so that the flexible electrode grafted with the cationic polymer is obtained.
The preparation method according to the invention, wherein the preparation method further comprises: and after mixing the polymer containing the nitrogen heterocyclic group and the cross-linking agent in a solvent, dip-coating the mixture on the surface of the flexible electrode with the self-assembled sensing layer to obtain the sensor with the diffusion limiting film.
ADVANTAGEOUS EFFECTS OF INVENTION
The self-assembled redox polymer of the invention can prepare the sensing layer of the sensor without introducing a cross-linking agent.
The self-assembled sensing layer of the sensor has no cross-linking agent, but the sensor still has excellent electrochemical performance, can respond to glucose concentration, and has high detection sensitivity, rapidness, accuracy, good stability and repeatability.
Furthermore, the preparation method of the sensor is simple and easy to implement, raw materials are easy to obtain, the prepared sensing layer does not need to introduce an additional small molecule cross-linking agent, the operation is easy, and the problem of cross-linking agent leakage can be avoided.
Drawings
Fig. 1 shows a cross-sectional view of the basic structure of a flexible electrode.
Fig. 2 shows a cross-sectional view of the basic structure of the sensor.
FIG. 3 shows a plot of sensor current signal versus time for a self-assembled sensing layer containing embodiment 2 of the present invention;
FIG. 4 shows a plot of non-self-assembled sensor current signal versus time for comparative example 1 of the present invention;
FIG. 5 shows a linear plot of a sensor comprising a self-assembled sensing layer of example 3 of the present invention;
fig. 6 shows a non-self-assembled sensor linearity map of comparative example 1 of the present invention.
Detailed Description
The following describes the present invention in detail. The following description of the technical features is based on the representative embodiments and specific examples of the present invention, but the present invention is not limited to these embodiments and specific examples. It should be noted that:
in the present specification, the numerical range indicated by the term "numerical value a to numerical value B" means a range including the end point numerical value A, B.
In the present specification, unless specifically stated otherwise, "a plurality" of "a plurality of" etc. means a numerical value of 2 or more.
In this specification, the terms "substantially", "substantially" or "substantially" mean that the error is less than 5%, or less than 3% or less than 1% compared to the relevant perfect or theoretical standard.
In the present specification, "%" means mass% unless otherwise specified.
In the present specification, the meaning of "can" includes both the meaning of performing a certain process and the meaning of not performing a certain process.
In this specification, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event occurs and instances where it does not.
Reference throughout this specification to "some specific/preferred embodiments," "other specific/preferred embodiments," "an embodiment," and so forth, means that a particular element (e.g., feature, structure, property, and/or characteristic) described in connection with the embodiment is included in at least one embodiment described herein, and may or may not be present in other embodiments. In addition, it is to be understood that the elements may be combined in any suitable manner in the various embodiments.
<First aspect>
A first aspect of the present invention provides a self-assembled redox polymer having the structure:
wherein R is 1 And R is 2 Each independently represents a bond, and R 1 And R is 2 The same or different;
m represents a transition metal;
L 1 、L 2 、L 3 、L 4 、L 5 and L 6 Each independently represents a ligand which is a heterocyclic ring and is coordinately bound to M through a heteroatom of the heterocyclic ring;
X represents a self-assembled group;
y represents an anion and is preferably selected from the group consisting of,
a represents an integer; b represents the number of anions and is a natural number; wherein b > a+1;
n, m and r represent natural numbers, and the sum of n, m and r is 20 or more, for example 40 or more;
e. g and f represent integers such as: all three can be integers of 0-20;
the invention prepares the sensing layer of the sensor by using self-assembled redox polymer without introducing cross-linking agent.
In some specific embodiments, for R 1 Or R is 2 It may be selected from- (CR) i R s )-、-O-、-S-、-C(O)O-、-S(O) 2 NR k -、-OC(O)NR m -、-OC(S)NR n -、-C(O)NR t -、-NR u -、-CR v ＝N-O-、-CR w ＝NNR x -and SiR y R z Wherein R is i And R is s Each independently is hydrogen, chlorine, fluorine, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, or an alkynyl group having 2 to 6 carbon atoms; r is R k 、R m 、R n 、R t 、R u 、R v 、R w 、R x 、R y And R is z Each independently is hydrogen, or a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms. The self-assembled groups are grafted onto the redox polymer by means of grafting by means of one or a combination of two or more of electrophilic, nucleophilic or condensation reactions, forming a self-assembled redox polymer.
The present invention is not particularly limited to M, and may be any feasible transition metal. In particular, the transition metal may be osmium, ruthenium, vanadium, cobalt or iron.
For L 1 、L 2 、L 3 、L 4 、L 5 And L 6 ，L 1 、L 2 、L 3 、L 4 、L 5 And L 6 Each independently is a monodentate ligand, or, L 1 、L 2 、L 3 、L 4 、L 5 And L 6 One of which is with L 1 、L 2 、L 3 、L 4 、L 5 Or L 6 The other ligands of (a) bind to form a multidentate ligand. In some specific embodiments, L 1 The heterocyclic ring of (2) is a nitrogen-containing heterocyclic ring, and is coordinately bonded to M through a nitrogen atom of the heterocyclic ring. Preferably, the nitrogen-containing heterocycle includes a substituted or unsubstituted pyridine, a substituted or unsubstituted imidazole, a substituted or unsubstituted 2,2 '-bipyridine, a substituted or unsubstituted 2- (2-pyridyl) imidazole, or a 2,2' -bipyridine.
The substituents on the above nitrogen-containing heterocycle are not particularly limited and may be any available substituents in the art, for example: it may be an inorganic group or an organic group having 1 to 10 carbon atoms, preferably an organic group having 1 to 5 carbon atoms. Preferably, as the inorganic group, it may be halogen, amino, hydroxyl, phosphate, phosphonate, metaphosphate, nitro, sulfate, sulfonate, cyano, thiocyano, mercapto, carbonate, phosphonate, or the like; as the organic group, it may be an alkyl group, an alkoxy group, a carboxyl group, a carboxyalkoxy group, a carbonate group, an alkyl ether group, an alkyl ester group, a thioether group, a thioester group, an acetal group, a carbamate group, an ureido group, an amide group, an imide group, a cycloalkyl group, a heterocyclic group, or the like. Wherein the alkyl groups can be straight-chain alkyl groups or branched-chain alkyl groups with the carbon number of 1-10, the alkoxy groups can be straight-chain alkoxy groups or branched-chain alkoxy groups with the carbon number of 1-10, and the cycloalkyl groups can be cycloalkyl groups with the carbon number of 3-10. Preferably, the halogen may be F, cl or Br atoms.
Further, in some specific embodiments, L 2 、L 3 、L 4 、L 5 And L 6 At least three of the heterocycles are the above nitrogen-containing heterocycles. Preferably L 1 、L 2 、L 3 、L 4 、L 5 And L 6 Each of the heterocyclic rings is the above-mentioned nitrogen-containing heterocyclic ring.
Further, in some specific embodiments, L 1 、L 2 、L 3 、L 4 、L 5 And L 6 Is combined to form at least one multidentate ligand. Preferably L 1 、L 2 、L 3 、L 4 、L 5 And L 6 Is bound to form at least two multidentate ligands.
Specifically, L 1 、L 2 、L 3 、L 4 、L 5 And L 6 Is combined to form at least two multidentate ligands selected from the group consisting of substituted and unsubstituted 2,2 'bipyridyl, substituted and unsubstituted 2- (2-pyridyl) imidazole, and substituted and unsubstituted 2,2' -bipyridyl. The present invention is not particularly limited as to the substituents, and may be any available ones in the art, for example: it may be an inorganic group or an organic group having 1 to 10 carbon atoms, preferably an organic group having 1 to 5 carbon atoms. Preferably, as the inorganic group, it may be halogen, amino, hydroxyl, phosphate, phosphonate, metaphosphate, nitro, sulfate, sulfonate, cyano, thiocyano, mercapto, carbonate, phosphonate, or the like; for organic groups, they may be alkyl, alkoxy, carboxyl Haloalkoxy, carbonate, alkyl ether, alkyl ester, thioether, thioester, acetal, carbamate, urea, amide, imide, cycloalkyl, heterocyclic, and the like. Wherein the alkyl groups can be straight-chain alkyl groups or branched-chain alkyl groups with the carbon number of 1-10, the alkoxy groups can be straight-chain alkoxy groups or branched-chain alkoxy groups with the carbon number of 1-10, and the cycloalkyl groups can be cycloalkyl groups with the carbon number of 3-10. Preferably, the halogen may be F, cl or Br atoms.
In some specific embodiments, the self-assembling group is selected from one of the following groups substituted with a substituent:
wherein c and d are integers from 1 to 16.
In particular, the substituents of the self-assembling groups may be selected from benzyl bromide, benzyl chloride, -F, -Cl, -Br, -I, -NO 2 、-COOH、-SO 3 H、-NH 2 -OH, an alkoxy group having 1 to 6 carbon atoms or an alkyl group having 1 to 6 carbon atoms.
The present invention is not particularly limited with respect to Y, and may be any anion practicable in the art, for example: halogen anions, cl - 、I - 、F - 、Br – Etc.
Further, in the present invention, the self-assembled redox polymer has a cleavage molecular weight of 1000Da or more.
The self-assembled redox polymer is obtained by grafting self-assembled groups on the redox polymer, the self-assembled redox polymer contains groups which are covalently bound with amino acid residues of glucose responsive enzyme, and an electron mediator in the self-assembled redox polymer can swing freely. The sensing layer of the sensor prepared by self-assembled redox polymer can realize the automatic combination with glucose responsive enzyme without a cross-linking agent.
<Second aspect>
In a second aspect, the present invention provides a method for preparing a self-assembled redox polymer according to the first aspect of the present invention, which comprises grafting self-assembled groups onto the redox polymer, preferably by a method comprising one or a combination of more than two of electrophilic reaction, nucleophilic reaction and condensation reaction.
Specifically, the preparation method of the self-assembled redox polymer comprises the following steps:
obtaining a redox polymer and a compound providing self-assembling groups,
mixing a redox polymer and a compound providing a self-assembled group in a solvent to obtain a mixed product,
And (3) carrying out grafting reaction on the mixed product to obtain a reaction product.
In some embodiments of the invention, the concentration of redox polymer in the mixed product is from 1mg/mL to 100mg/mL, for example: 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, etc., the concentration of the compound providing the self-assembled group is 1mg/mL to 50mg/mL, for example: 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, etc. The mixing time is not particularly limited, and may be set as needed, for example: 12-24 hours, for example: 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, etc.
The solvent is not particularly limited, and may be a solvent commonly used in the art, preferably an organic solvent, for example: one or more of N, N-Dimethylformamide (DMF), ethanol, methanol, N-dimethylacetamide and N-methylpyrrolidone.
In some specific embodiments, the grafting means comprises one or a combination of two or more of electrophilic, nucleophilic, and condensation reactions.
In some embodiments, the self-assembling group-providing compound may be an alkyl boric acid having 1 to 16 carbon atoms, preferably 1 to 6 carbon atoms 1-16, preferably 1-6, alkyl aldehyde, phenylboronic acid, benzaldehyde, benzyl alcohol, catechol, acyl chloride or anhydride, each of which may be substituted with a substituent selected from benzyl bromide, benzyl chloride, -F, -Cl, -Br, -I and NO 2 、-COOH、-SO 3 H、-NH 2 -OH, an alkoxy group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms, a halogen-substituted alkyl group having 1 to 6 carbon atoms, etc.; such as dopamine, 3, 4-dihydroxyphenylalanine, 4-bromomethylphenylboronic acid, etc.
The present invention is not particularly limited with respect to specific reaction conditions, and the reaction may be carried out according to electrophilic reaction, nucleophilic reaction, condensation reaction, to obtain a desired reaction product.
Further, in order to allow the obtained reaction product to be soluble in water, in the form of ions, an anion exchange resin may be added to the reaction product, thereby obtaining a solution containing a self-assembled redox polymer. The amount of the anion exchange resin is not particularly limited, and the anion exchange resin may be added as required, and the time for the addition may be generally 5 to 20 hours, for example: 7 hours, 9 hours, 10 hours, 12 hours. 14 hours, 16 hours, 18 hours, etc.
Further, filtering with ultrafiltration membrane to obtain self-assembled redox polymer solution with molecular weight to be cut, purifying to waste liquid conductivity less than or equal to 50 μs/cm, lyophilizing, and cutting self-assembled redox polymer with molecular weight above 1000 Da.
<Third aspect of the invention>
As shown in fig. 2: a third aspect of the invention provides a sensor comprising:
a flexible electrode comprising a substrate, a working electrode, a counter electrode, and a reference electrode, wherein,
the working electrode and the counter electrode are respectively arranged at two sides of the base material, and a dielectric layer is arranged at one side of the counter electrode far away from the base material;
the side of the working electrode far away from the base material is provided with the reference electrode, a dielectric layer is arranged between the working electrode and the reference electrode, the side of the reference electrode far away from the working electrode is provided with the dielectric layer, and,
the surface of the working electrode has an exposed portion without a dielectric layer and a reference electrode, and the surface of the exposed portion is loaded with a self-assembled sensing layer (sensing layer in fig. 2) derived from the self-assembled redox polymer and redox enzyme according to the first aspect of the present invention.
The self-assembled redox polymer of the present invention contains groups covalently bound to the amino acid residues of the redox enzyme, while the electron mediator in the self-assembled redox polymer can swing freely, and the self-assembled sensing layer of the present invention does not contain a cross-linking agent.
In some specific embodiments, the mass ratio of self-assembling redox polymer to redox enzyme is from 1:0.5 to 1:2, for example: 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, etc. When the mass ratio of the self-assembled redox polymer and the redox enzyme is 1:0.5 to 1:2, a self-assembled sensing layer excellent in performance can be obtained.
Further, for the oxidoreductase, it may be a glucose responsive enzyme such as: glucose oxidase or glucose dehydrogenase, preferably glucose oxidase.
The materials of the working electrode, the counter electrode and the reference electrode of the present invention are not particularly limited, and may be those commonly used in the art. Specifically, the conductive materials of the working electrode, the counter electrode and the reference electrode may be one or a combination of more than two of graphite, graphene, nano carbon, carbon nano tube, silver/silver chloride and the like.
In addition, in the present invention, the working, counter and reference electrodes can be formed using screen printed conductive material on the substrate.
Further, in the present invention, the substrate may be a flexible film, for example: polyethylene terephthalate (PET), and the like.
Further, in the present invention, the thicknesses of the working electrode, the counter electrode, and the reference electrode are not particularly limited, and may be set as needed. When screen printing a conductive material on a substrate to prepare a working electrode, a counter electrode and a reference electrode, the thickness of the printed conductive material may be 1-100 μm, and when directly using a conductive material as the working electrode, the counter electrode and the reference electrode, the thickness of the working electrode, the counter electrode and the reference electrode may be 1-100 μm as well.
The dielectric layer, i.e., the insulating layer, of the present invention is not particularly limited, and the material of the dielectric layer may be any commercially available insulating material. The thickness of the dielectric layer is not particularly limited and may be 1 to 50. Mu.m.
In some specific embodiments, the surface of the flexible electrode has a cationic polymer; preferably, the weight average molecular weight of the cationic polymer may be in the range 1000-50000Da.
In other specific embodiments, a diffusion limiting membrane is also provided on the surface of the sensor. The diffusion limiting membrane is used for limiting the flow of glucose to the working electrode in the electrochemical sensor, so that the linear response range and the stability of the sensor are improved. And the diffusion-limiting membrane may serve multiple functions such as biocompatibility, tamper resistance, etc.
Further, in the present invention, the diffusion-limiting membrane is derived from a polymer containing a nitrogen-containing heterocyclic group and a crosslinking agent; preferably, the mass ratio of the polymer containing nitrogen heterocyclic groups to the cross-linking agent is 64:1 to 10:1, for example: 60:1, 55:1, 50:1, 45:1, 40:1, 35:1, 30:1, 25:1, 20:1, 15:1, etc. The mass ratio of the polymer containing the nitrogen heterocyclic group to the cross-linking agent is 64:1 to 10:1, the function of the diffusion barrier is limited to be most effectively exerted.
Specifically, the polymer containing nitrogen heterocyclic groups can be one or more than two of polyvinyl pyridine, polyvinyl pyridine-styrene copolymer, polyvinyl imidazole and the like, and the cross-linking agent comprises one or more than two of poly (dimethylsiloxane) -diglycidyl ether, polyethylene glycol diglycidyl ether, P- (2, 3-epoxypropoxy) -N, N-bis (2, 3-epoxypropoxy) aniline and diimine adipic acid dimethyl ester dihydrochloride.
Further, the polymer containing nitrogen heterocyclic groups is modified with hydrophilic groups. The hydrophilic group can be one or more than two of polyethylene glycol group, sulfonic acid group, amino group and carboxyl group, and is used for improving the biocompatibility of the diffusion limiting layer.
<Fourth aspect of>
A fourth aspect of the present invention provides a method for manufacturing a sensor according to the third aspect of the present invention, comprising the steps of:
a step of preparing a flexible electrode, wherein the flexible electrode comprises a substrate, a working electrode, a counter electrode and a reference electrode;
and loading the self-assembled sensing layer on the exposed part of the working electrode, which is not provided with the dielectric layer and the reference electrode.
< preparation of Flexible electrode >
The invention employs a manner of stacking a working electrode, a counter electrode, and a reference electrode to prepare the flexible electrode.
Specifically, electrode substrate materials are taken, and conductive materials are printed on the surfaces of the electrode substrate materials to serve as conductive layers so as to prepare a working electrode and a counter electrode; the electrode substrate material can be a flexible polymer film, namely polyethylene terephthalate.
For example, a flexible polymer film may be used as a substrate, and a working electrode and a counter electrode are respectively disposed on two sides of the substrate, and then a dielectric layer is disposed on a side of the counter electrode away from the substrate; a dielectric layer is arranged on one side of the working electrode far away from the base material, a layer of conductive material is printed on one side of the dielectric layer far away from the working electrode to serve as a reference electrode, the required flexible electrode is obtained after laser cutting, and the surface of the working electrode is provided with a bare part without the dielectric layer and the reference electrode.
< preparation of sensor >
In the present invention, in view of maximizing the effect of the sensor, in some specific embodiments, plasma is used to initiate grafting of the cationic polymer to the surface of the flexible electrode, resulting in a pretreated flexible electrode. During the initiation of grafting, the gas flow rate of the plasma is between 10 mL/min and 500 mL/min, for example: 50 mL/min, 100 mL/min, 150 mL/min, 200 mL/min, 250 mL/min, 300 mL/min, 350 mL/min, 400 mL/min, 450 mL/min, etc.; the treatment power to initiate grafting is 30w-200w, for example: 50w, 80w, 100w, 120w, 150w, 180w, etc.; the treatment time to initiate grafting is 1 minute to 30 minutes, for example: 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, etc.
Specifically, the plasma includes one or a combination of two or more of argon, oxygen, hydrogen, nitrogen, or air. The cationic polymer comprises one or more than two of polyallylamine, poly (L-lysine), polyethyleneimine and polyamide-amine dendrimer.
Further, the self-assembled redox polymer and the redox enzyme are mixed in a buffer solution, wherein the concentration of the self-assembled redox polymer is 1mg/mL to 200mg/mL, for example: 20mg/mL, 50mg/mL, 80mg/mL, 100mg/mL, 120mg/mL, 150mg/mL, 180mg/mL, etc.; the concentration of oxidoreductase is 1mg/mL-200mg/mL such as: 20mg/mL, 50mg/mL, 80mg/mL, 100mg/mL, 120mg/mL, 150mg/mL, 180mg/mL, etc. The mixing time is not particularly limited, and may be set as needed, for example: 30 minutes to 200 minutes, for example: 50 minutes, 80 minutes, 100 minutes, 120 minutes, 150 minutes, 180 minutes, etc.
The buffer solution is not particularly limited, and may be a solvent commonly used in the art, preferably a buffer solution having a pH of 4.5 to 9, specifically, pH values of 5, 6, 7, 8, etc., for example: one or a combination of two or more of HEPES buffer solution, TES buffer solution, MES buffer solution, etc.
And after mixing the self-assembled redox polymer and the redox enzyme, loading the self-assembled redox polymer and the redox enzyme on the surface of the exposed part of the working electrode of the pretreated flexible electrode, which is not provided with the dielectric layer and the reference electrode. Specifically, a microsyringe is used for quantitatively dripping the sensing layer solution to the exposed part, and the flexible electrode with the self-assembled sensing layer is obtained after the flexible electrode is placed for 0.5 to 2 days at room temperature.
In some specific embodiments, the polymer containing nitrogen heterocyclic groups and the cross-linking agent are mixed in an alcohol buffer to obtain a mixed product; the mixed product was dip-coated on the surface of the flexible electrode, thereby obtaining a sensor having a diffusion-limiting film.
Specifically, the concentration of the polymer containing nitrogen heterocyclic groups in the mixed product is 1mg/mL-200mg/mL, for example: 20mg/mL, 50mg/mL, 80mg/mL, 100mg/mL, 120mg/mL, 150mg/mL, 180mg/mL, etc.; the concentration of the crosslinking agent is 0.5mg/mL-10mg/mL, for example: 0.8mg/mL, 1mg/mL, 3mg/mL, 5mg/mL, 7mg/mL, 9mg/mL, etc. The mixing time is not particularly limited, and may be set as needed.
The alcohol buffer is not particularly limited, and may be an alcohol buffer commonly used in the art, specifically, the alcohol buffer may be a mixture of an alcohol solvent and a buffer solution, preferably, the volume ratio of the alcohol solution to the buffer solution is 1-10:1, for example: 2:1, 4:1, 6:1, 8:1, etc. For the alcohol solvents, common solvents such as methanol, ethanol, propanol, etc., and for the buffer solution, a buffer solution having a pH of 4.5 to 9 is preferable, for example: one or a combination of two or more of HEPES buffer solution, TES buffer solution or MES buffer solution.
And dip-coating the mixed product on the surface of the flexible electrode, and curing to obtain the sensor with the diffusion limiting film. Specifically, the invention is not particularly limited as to the manner of dip coating, and in view of the applicability of the raw materials, a manner of dip coating a plurality of times, for example, 1 to 10 times, may be adopted, and the time interval may be 5 to 20 minutes each time. After dip coating, the mixed product was cured at room temperature for 1-2 days to give a sensor with diffusion limiting membrane.
Examples
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the examples, the compound of formula (I) used has the formula:
the preparation method of the compound shown in the formula (I) comprises the following steps:
in a 250mL single-port flask, 10g of polyvinylpyridine (mw= 160000,sigma aldrich) was dissolved in DMF (100 mL), 6-bromohexanoic acid (2.8 g) was added, nitrogen was replaced three times, the reaction solution was reacted at 90 ℃ for 24 hours, the reaction solution was cooled to room temperature, added dropwise to 600mL of ethyl acetate, and suction filtration and vacuum drying were performed to obtain 12g of caproic acid grafted polyvinylpyridine.
To a 250mL three-necked flask, 6-methyl-2-pyridinecarboxaldehyde (13 g), glyoxal (40%, 15 mL) and absolute ethanol (25 mL) were sequentially added, concentrated aqueous ammonia (41.6 mL) was slowly added dropwise under ice bath, and the mixture was stirred at room temperature for 24 hours, followed by purification to obtain 8.4g of 2- (6-methyl-2-pyridineimidazole) (yellow solid).
To a 500mL single-necked flask, 8.4g of 2- (6-methyl-2-pyridine) imidazole, 23g N-Boc-6-bromohexylamine, 125mL of DMF and 34.4g of anhydrous cesium carbonate were sequentially added, nitrogen was replaced for 3 times, the reaction was allowed to react at 90℃for 2 days, the reaction solution was cooled to room temperature, and 11.2g of 2- (6-methyl-2-pyridine) -1- (6-Boc-aminohexyl) imidazole was obtained by separation and purification.
To a 250mL single flask, 11g of 2- (6-methyl-2-pyridine) -1- (6-Boc-aminohexyl) imidazole and 55mL of ethanol were sequentially added, 4N HCl-dioxane (55 mL) was added dropwise at room temperature, and the reaction was carried out overnight at room temperature, filtered and dried under vacuum to give 9.9g of 2- (6-methyl-2-pyridine) -1- (6-aminohexyl) imidazole hydrochloride.
10g of biimidazole and 150mL of DMF are sequentially added into a 500mL single-port bottle, 6.28g of NaH (60%) is added in batches under ice bath, after the addition is completed, the mixture is stirred at room temperature for 0.5 hour, 31.93g of methyl p-toluenesulfonate is weighed and dissolved in 50mL of DMF, a solution of methyl p-toluenesulfonate is dropwise added under ice bath, after the dropwise addition is completed, and the reaction is stirred at room temperature overnight. The reaction solution was separated and purified to obtain 11.3g of 1,1 '-dimethyl-2, 2' -biimidazole (white solid).
Sequentially adding 1g K into 250mL three-necked flask 2 OsCl 6 0.67g of 1,1 '-dimethyl-2, 2' -biimidazole, 1g of lithium chloride and 40mL of ethylene glycol, replacing nitrogen gas for 3 times, reacting at 140 ℃ for 24 hours, cooling the reaction liquid to room temperature, adding 2- (6-methyl-2-pyridine) -1- (6-aminohexyl) imidazole hydrochloride, replacing nitrogen gas for three times, stirring the reaction liquid at 170 ℃ for 8 hours, cooling the reaction liquid to room temperature, separating and purifying to obtain 2.95g of Os (1, 1 '-dimethyl-2, 2' -biimidazole) 2 [2- (6-methyl-2-pyridine) -1- (6-aminohexyl) imidazole](PF 6 ) 3 。
Into a 50mL three-necked flask, sequentially added were polyvinylpyridine grafted with caproic acid (515 mg), DMF (10 mL), TSTU (202 mg) and DIEA (200 uL), stirred at room temperature for 7 hours, and Os (1, 1 '-dimethyl-2, 2' -biimidazole) was added in portions under ice bath 2 [2- (6-methyl-2-pyridine) -1- (6-aminohexyl) imidazole ](PF 6 ) 3 After the addition, stirring was carried out at room temperature for 24 hours. The reaction mixture was added dropwise to 200mL of ethyl acetate, and the mixture was filtered, and the solid was isolated and purified to obtain 745mg of a compound of formula (I) (dark blue solid).
Example 1
Synthesis of self-assembled redox polymer a: 100mg of the compound represented by formula (I), 9.08mg of 4-bromomethylbenzoic acid and 2mL of anhydrous DMF are sequentially added into a 25mL single-port bottle, the mixture is stirred overnight at room temperature in a nitrogen atmosphere, 50mL of pure water is added into the reaction solution, 2g of chloride ion exchange resin is added into the reaction solution, the mixture is stirred for 10 hours, the mixture is filtered, the water phase is purified to the conductivity of the waste liquid being less than or equal to 50 mu s/cm by using an ultrafiltration membrane with the model of 5000Da (namely, the cutting molecular weight of the product is more than 5000 Da), and the waste liquid is freeze-dried, so that 50mg of yellow-green solid is obtained, and the structure is shown as follows.
Preparation of a flexible electrode: the flexible electrode is prepared by stacking the electrodes.
Taking three electrode substrate materials, and printing carbon slurry on the surfaces of the three electrode substrate materials as a conductive layer to prepare a working electrode and a counter electrode; the electrode substrate material is a flexible polymer film, namely polyethylene terephthalate.
Taking one of the flexible polymer films as a base material, respectively arranging a working electrode and a counter electrode on two sides of the base material, and then arranging a dielectric layer on one side of the counter electrode far away from the base material; a dielectric layer is sequentially arranged on one side of the working electrode, which is far away from the base material, a layer of silver/silver chloride slurry is printed on one side of the dielectric layer, which is far away from the working electrode, and is used as a reference electrode, and the required flexible electrode is obtained after laser cutting, and the flexible electrode is shown in a specific view in fig. 1. Wherein the working and counter electrodes are printed with a carbon layer thickness of 15 μm, the reference electrode is 20 μm and the dielectric layer is 10 μm.
Pretreatment of flexible electrode: after the flexible electrode was treated with (100 mL/min) air plasma (80 w,5 min), the treated flexible electrode was immersed in 15mg/mL poly (L-lysine) (weight average molecular weight: 15000-30000 Da) overnight at 37 ℃. Washing with water for three times, and airing to obtain the flexible electrode grafted with the cationic polymer.
Preparation of a flexible electrode containing a self-assembled sensing layer a: 3.4mg/mL glucose oxidase and 43mg/mL self-assembled redox polymer a were mixed in a volume ratio of 10:1, vortexed for 3min, and the solvent was 10mmol/L HEPES buffer solution at pH 8. Spotting 20nL of sensing layer solution on exposed part of working electrode of flexible electrode grafted with cationic polymer by using microinjector, and its effective area is 1mm 2 Curing for one day at room temperature to obtain the flexible electrode containing the self-assembled sensing layer a.
Preparation of limiting diffusion film: the diffusion limiting solution was formulated from a sulfonic acid group modified polyvinyl pyridine-styrene copolymer with polyethylene glycol diglycidyl ether (mw=200 Da) solution. Wherein the solvent used for dissolving the polyethylene glycol diglycidyl ether (mw=200da) is ethanol and 10mmol/L HEPES buffer with pH of 8, wherein the volume ratio of ethanol to HEPES buffer is 4:1; the concentration of the sulfonic acid group modified polyvinyl pyridine-styrene copolymer and the polyethylene glycol diglycidyl ether in the limiting diffusion solution was 64mg/mL and 2mg/mL, respectively. Dip-coating the flexible electrode containing the self-assembled sensing layer a for 6 times by using a diffusion limiting solution at intervals of 10min each time, and curing for 1 day at room temperature after the dip-coating is finished, so that the required sensor a is obtained, and the structure diagram is shown in figure 2.
Example 2
Synthesis of self-assembled redox polymer b: 100mg of non-self-assembled redox polymer, 50mg of 4-bromomethylbenzaldehyde and 2mL of anhydrous DMF are sequentially added into a 25mL single-port bottle, the mixture is stirred for 24 hours at room temperature in a nitrogen atmosphere, 50mL of pure water is added into the reaction solution, 3g of chloride ion exchange resin is added into the reaction solution, the mixture is stirred for 10 hours, the mixture is filtered, the water phase is purified to the conductivity of the waste liquid being less than or equal to 50us/cm by an ultrafiltration membrane with the model of 30000Da (namely, the cutting molecular weight of the product is more than 30000 Da), and the waste liquid is freeze-dried, so that 68mg of yellowish green solid is obtained.
Preparation of a flexible electrode: the flexible electrode is prepared by stacking the electrodes.
Taking three electrode substrate materials, and printing carbon slurry on the surfaces of the three electrode substrate materials as a conductive layer to prepare a working electrode and a counter electrode; the electrode substrate material is a flexible polymer film, namely polyethylene terephthalate.
Taking one of the flexible polymer films as a base material, respectively arranging a working electrode and a counter electrode on two sides of the base material, and then arranging a dielectric layer on one side of the counter electrode far away from the base material; a dielectric layer is sequentially arranged on one side of the working electrode, which is far away from the base material, a layer of silver/silver chloride slurry is printed on one side of the dielectric layer, which is far away from the working electrode, and is used as a reference electrode, and the required flexible electrode is obtained after laser cutting, and the flexible electrode is shown in a specific view in fig. 1. Wherein the thickness of the carbon layer printed on the working electrode and the counter electrode is 15 μm, the thickness of the reference electrode is 20 μm, and the thickness of the dielectric layer is 10 μm.
Pretreatment of flexible electrode: after the flexible electrode was treated with (100 mL/min) air plasma (80 w,5 min), the treated flexible electrode was immersed in 15mg/mL poly (L-lysine) (weight average molecular weight: 15000-30000 Da) overnight at 37 ℃. Washing with water for three times, and airing to obtain the flexible electrode grafted with the cationic polymer.
Flexible electrode containing self-assembled sensing layer b: 20mg/mL glucose oxidase and 20mg/mL self-assembled redox polymer b were mixed in a volume ratio of 1:2, vortexed for 3min, and the solvent was 10mmol/L HEPES buffer solution at pH 8. Spotting 20nL of sensing layer solution on exposed part of working electrode of flexible electrode grafted with cationic polymer by using microinjector, and its effective area is 1mm 2 Curing for one day at room temperature to obtain the flexible electrode containing the self-assembled sensing layer b.
Preparation of limiting diffusion film: the diffusion limiting solution was formulated from a sulfonic acid group modified polyvinyl pyridine-styrene copolymer with polyethylene glycol diglycidyl ether (mw=200 Da) solution. Wherein the solvent used to dissolve polyethylene glycol diglycidyl ether (mw=200da) is ethanol mixed with 10mmol/L HEPES buffer at pH 8, wherein ethanol: the volume ratio of HEPES buffer solution is 4:1; the concentration of the sulfonic acid group modified polyvinyl pyridine-styrene copolymer and the polyethylene glycol diglycidyl ether in the limiting diffusion solution was 64mg/mL and 2mg/mL, respectively. Dip-coating the flexible electrode containing the self-assembled sensing layer b for 6 times by using a diffusion limiting solution at intervals of 10min each time, and curing for 1 day at room temperature after the dip-coating is finished, so that the required sensor b is obtained, and the structure diagram is shown in figure 2.
Example 3
Preparation of a flexible electrode: the flexible electrode is prepared by stacking the electrodes.
Taking three electrode substrate materials, and printing carbon slurry on the surfaces of the three electrode substrate materials as a conductive layer to prepare a working electrode and a counter electrode; the electrode substrate material is a flexible polymer film, namely polyethylene terephthalate.
Taking one of the flexible polymer films as a base material, respectively arranging a working electrode and a counter electrode on two sides of the base material, and then arranging a dielectric layer on one side of the counter electrode far away from the base material; a dielectric layer is sequentially arranged on one side of the working electrode, which is far away from the base material, a layer of silver/silver chloride slurry is printed on one side of the dielectric layer, which is far away from the working electrode, and is used as a reference electrode, and the required flexible electrode is obtained after laser cutting, and the flexible electrode is shown in a specific view in fig. 1. Wherein the thickness of the carbon layer printed on the working electrode and the counter electrode is 15 μm, the thickness of the reference electrode is 20 μm, and the thickness of the dielectric layer is 10 μm.
Pretreatment of flexible electrode: after the flexible electrode was treated with (100 mL/min) nitrogen plasma (80 w, 5 min), the treated flexible electrode was immersed in 15mg/mL poly (L-lysine) (weight average molecular weight: 15000-30000 Da) overnight at 37 ℃. Washing with water for three times, and airing to obtain the flexible electrode grafted with the cationic polymer.
Preparation of a flexible electrode containing a self-assembled sensing layer c: 20mg/mL glucose oxidase was mixed with 20mg/mL self-assembled redox polymer b of example 2 at a volume ratio of 1:2, vortexed for 3min, and the solvent was 10mmol/L MES buffer solution at pH 5.5. Spotting 20nL of sensing layer solution on exposed part of working electrode of flexible electrode grafted with cationic polymer by using microinjector, and effective area is 1mm 2 Curing for one day at room temperature to obtain the flexible electrode containing the self-assembled sensing layer c.
Preparation of limiting diffusion film: the diffusion limiting solution is prepared from a polyvinylpyridine-styrene copolymer modified by sulfonic groups and a P- (2, 3-epoxypropoxy) -N, N-di (2, 3-epoxypropyl) aniline solution. Wherein, the solvent used for dissolving the P- (2, 3-epoxypropoxy) -N, N-di (2, 3-epoxypropyl) aniline is ethanol and 10mmol/L HEPES buffer solution with pH value of 8, wherein, the volume ratio of the ethanol to the HEPES buffer solution is 4:1; the concentration of the sulfonic acid group modified polyvinyl pyridine-styrene copolymer and the P- (2, 3-glycidoxy) -N, N-bis (2, 3-epoxypropyl) aniline solution in the limiting diffusion solution were 64mg/mL and 2mg/mL, respectively. Dip-coating the flexible electrode containing the self-assembled sensing layer c for 6 times by using a diffusion limiting solution at intervals of 10min each time, and curing for 1 day at room temperature after the dip-coating is finished, so that the required sensor c is obtained, and the structure diagram is shown in figure 2.
Comparative example 1
Preparation of a flexible electrode: the flexible electrode is prepared by stacking the electrodes.
Taking three electrode substrate materials, and printing carbon slurry on the surfaces of the three electrode substrate materials as a conductive layer to prepare a working electrode and a counter electrode; the electrode substrate material is a flexible polymer film, namely polyethylene terephthalate.
Taking one of the flexible polymer films as a base material, respectively arranging a working electrode and a counter electrode on two sides of the base material, and then arranging a dielectric layer on one side of the counter electrode far away from the base material; a dielectric layer is sequentially arranged on one side of the working electrode, which is far away from the base material, a layer of silver/silver chloride slurry is printed on one side of the dielectric layer, which is far away from the working electrode, and is used as a reference electrode, and the required flexible electrode is obtained after laser cutting, and the flexible electrode is shown in a specific view in fig. 1. Wherein the thickness of the carbon layer printed on the working electrode and the counter electrode is 15 μm, the thickness of the reference electrode is 20 μm, and the thickness of the dielectric layer is 10 μm.
Pretreatment of flexible electrode: after the flexible electrode was treated with (100 mL/min) air plasma (80 w, 5 min), the treated flexible electrode was immersed in 15mg/mL poly (L-lysine) (weight average molecular weight: 15000-30000 Da) overnight at 37 ℃. Washing with water for three times, and airing to obtain the flexible electrode grafted with the cationic polymer.
Preparation of a non-self-assembled sensing layer: 3.4mg/mL of glucose oxidase and 43mg/mL of the compound represented by formula (I) were mixed at a volume ratio of 10:1, vortexed for 3min, and the solvent was 10mmol/L HEPES buffer solution at pH 8. Spotting 20nL of sensing layer solution on exposed part of working electrode of flexible electrode grafted with cationic polymer by using microinjector, and its effective area is 1mm 2 Curing for one day at room temperature to obtain the flexible electrode containing the non-self-assembled sensing layer.
Preparation of limiting diffusion film: the diffusion limiting solution is prepared from a polyvinylpyridine-styrene copolymer modified by sulfonic acid groups and a polyethylene glycol diglycidyl ether (MW=200Da) solution. Wherein the solvent used for dissolving the polyethylene glycol diglycidyl ether (mw=200da) is ethanol and 10mmol/L HEPES buffer solution with pH of 8, wherein the volume ratio of ethanol to HEPES buffer solution is 4:1; the concentration of the sulfonic acid group modified polyvinyl pyridine-styrene copolymer and the polyethylene glycol diglycidyl ether in the limiting diffusion solution was 64mg/mL and 2mg/mL, respectively. Dip-coating the flexible electrode containing the non-self-assembled sensing layer for 6 times by using a diffusion limiting solution, wherein each time is 10min, and after the dip-coating is finished, curing for 1 day at room temperature, thus obtaining the required non-self-assembled sensor.
Performance testing
1. The sensor implant (5 mM) was immersed in a clear sample bottle containing 10mM glucose solution, and the solution was maintained at a constant temperature of 37 ℃. And (3) performing stability test by adopting a timing current method (i-t), and testing the curve of the current signal changing along with time. Fig. 3 is a graph of the time-dependent sensor current signal of example 2 including a self-assembled sensing layer, and fig. 4 is a graph of the time-dependent sensor current signal of comparative example 1. As can be seen from fig. 3 and 4, the self-assembled sensor was continuously tested for 14 days with a current decay of < 20%, whereas the non-self-assembled sensor of comparative example 1 had no current signal.
The present application conducted the above-described related tests for example 1 and example 3, and the results were substantially the same as those of example 2.
2. Sensor linearity test
The sensor-implanted portion (5 mm) was immersed in a flask containing a PBS solution and the flask was placed on a magnetic stirrer bench, and the solution was maintained at 37℃by an oil bath and maintained at a rotational speed of 120 revolutions per minute. A linear test was performed using chronoamperometry (i-t). Sensor testing the flask was preheated for about 15min and a high concentration of glucose solution was injected into the flask at regular intervals to continuously increase the glucose concentration in the flask from 0-30mM, and the results are shown in FIGS. 5 and 6. Fig. 5 is a linear plot of the sensor containing the self-assembled sensing layer of example 3, and fig. 6 is a non-self-assembled sensor linear plot of comparative example 1. As shown in fig. 5 and 6, the self-assembled sensor showed excellent linearity at a concentration of 0-30mmol of glucose solution, whereas the non-self-assembled sensor of comparative example 1 had no linearity.
The present application conducted the above-described related tests on examples 1 and 2, and the results were substantially the same as those of example 3.
It should be noted that, although the technical solution of the present application is described in specific examples, those skilled in the art can understand that the present application should not be limited thereto.
The foregoing description of embodiments of the application has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described. The terminology used herein was chosen in order to best explain the principles of the embodiments, the practical application, or the technical improvement in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

Claims (15)

1. A self-assembled redox polymer characterized by the following structure:
wherein R is 1 And R is 2 Each independently represents a bond, and R 1 And R is 2 The same or different;
m represents a transition metal;
L 1 、L 2 、L 3 、L 4 、L 5 and L 6 Each independently represents a ligand which is a heterocyclic ring and is coordinately bound to M through a heteroatom of the heterocyclic ring;
X represents a self-assembled group;
y represents an anion and is preferably selected from the group consisting of,
a represents an integer; b represents the number of anions and is a natural number; wherein b > a+1;
n, m and r represent natural numbers, and the sum of n, m and r is more than 20;
e. g and f represent integers;
the self-assembled group is selected from one of the following groups substituted or unsubstituted by a substituent:
wherein c and d are integers from 1 to 16;
the substituent of the self-assembly group is selected from benzyl bromide, benzyl chloride, -F, -Cl, -Br, -I and-NO 2 、-COOH、-SO 3 H、-NH 2 -OH, an alkoxy group having 1 to 6 carbon atoms or an alkyl group having 1 to 6 carbon atoms.
2. The self-assembling redox polymer of claim 1, wherein the self-assembling redox polymer has a cleavage molecular weight of 1000Da or more.
3. A method of preparing a self-assembled redox polymer according to claim 1 or claim 2, comprising grafting self-assembled groups onto the redox polymer.
4. The method according to claim 3, wherein the grafting method comprises one or a combination of more than two of electrophilic reaction, nucleophilic reaction and condensation reaction.
5. A sensor, comprising: a flexible electrode comprising a substrate, a working electrode, a counter electrode, and a reference electrode, wherein,
The working electrode and the counter electrode are respectively arranged at two sides of the base material, and a dielectric layer is arranged at one side of the counter electrode far away from the base material;
the side of the working electrode far away from the base material is provided with the reference electrode, a dielectric layer is arranged between the working electrode and the reference electrode, the side of the reference electrode far away from the working electrode is provided with the dielectric layer, and,
the surface of the working electrode has an exposed portion where the dielectric layer and the reference electrode are not provided, and a self-assembled sensing layer derived from the self-assembled redox polymer and the redox enzyme according to claim 1 or 2 is loaded on the surface of the exposed portion.
6. The sensor of claim 5, wherein the mass ratio of self-assembling redox polymer to redox enzyme is 1:0.5 to 1:2.
7. The sensor of claim 5 or 6, wherein the surface of the flexible electrode has a cationic polymer.
8. The sensor of claim 7, wherein the cationic polymer has a weight average molecular weight of 1000-50000Da.
9. A sensor according to claim 5 or 6, characterized in that a diffusion limiting membrane is further provided on the surface of the flexible electrode with the self-assembled sensing layer.
10. The sensor of claim 9, wherein the diffusion limiting membrane is derived from a polymer comprising a nitrogen-containing heterocyclic group and a cross-linking agent.
11. The sensor of claim 10, wherein the polymer of nitrogen-containing heterocyclic groups is modified with hydrophilic groups.
12. A method of manufacturing a sensor according to any one of claims 5 to 11, comprising the steps of:
a step of preparing a flexible electrode, wherein the flexible electrode comprises a substrate, a working electrode, a counter electrode and a reference electrode;
and loading the self-assembled sensing layer on the exposed part of the working electrode, which is not provided with the dielectric layer and the reference electrode.
13. The method according to claim 12, wherein the self-assembled redox polymer and the redox enzyme are mixed and then carried on the surface of the exposed portion of the working electrode where the dielectric layer and the reference electrode are not provided, thereby obtaining the flexible electrode having the self-assembled sensing layer.
14. The method of claim 12 or 13, wherein the grafting cationic polymer is initiated by plasma on the surface of the flexible electrode before loading the self-assembled sensing layer, resulting in a grafted cationic polymer flexible electrode.
15. The production method according to claim 12 or 13, characterized in that the production method further comprises: after mixing the polymer containing the nitrogen heterocyclic group and the cross-linking agent in a solvent, dip-coating the mixture on the surface of the flexible electrode with the self-assembled sensing layer to obtain the sensor with the diffusion limiting film.
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